Numerous techniques are available to the skilled practitioner for detecting or monitoring hydrogen peroxide present as a reactant or treating agent in a chemical process or which represents an analyte in a diagnostic protocol. The hydrogen peroxide that is to be monitored may be added directly into the system and its depletion subsequently monitored, or it may be formed from the systems' reactants, with or without catalysts.
Hydrogen peroxide is used and monitored in metal extraction processes; decomposition of alkyl phosphates; synthesis of sulfonic acids and carboxylic acid ester derivatives thereof, oxidation processes, and sterilization systems. In the diagnostic area, hydrogen peroxide is often used to indirectly indicate the presence or absence of an analyte of interest. Such analytes include, but are not limited to, cholesterol, glucose, uric acid, ascorbic acid, triglycerides, and thiol compounds.
Prior methods for the detection and quantitation of such peroxide-forming analytes were calorimetric and/or spectrophotometric (Allain, C. C., et. al., cf. cholesterol reprint). For example, in U.S. Pat. No. 4,238,195 (Boguslaski, et al.) the excitation of a label is preferably accomplished by exposure to a substance, such as a high energy intermediate produced by the reaction between hydrogen peroxide and highly reactive materials such as oxalyl chloride, oximides and bis-oxalate esters. The mechanism of the Boguslaski system would appear to result from the reaction of hydrogen peroxide and various reactive bis-oxalate esters to form a high energy intermediate believed to be, for example, 1,2-dioxethanedione or activated carbon dioxide. The high energy intermediate then transfers energy to a fluorescer which thereafter emits light with a spectrum essentially similar to its normal fluorescent spectrum. The electromagnetic radiation released can be in the infra red, visible, or ultraviolet range, and the fluorescer-label is neither a consumable reactant nor is altered chemically in any way during the reaction.
U.S. Pat. No. 4,372,745 to Mandle, et al. relates to chemical luminescence amplification substrate systems for amino acid chemistry involving microencapsulated fluorescers. Mandle discusses prior art techniques where hydrogen peroxide, an essential component in many chemiluminescent reactions, is the specie selected for use in detecting the analyte of interest. For example, Mandle discusses the prior art oxidation of glucose with glucose oxidase as a source of H.sub.2 O.sub.2 which, in turn, is reacted with luminol to produce chemiluminescence in proportion to the initial glucose concentration. However, a limit of detection of 10.sup.-8 M peroxide is obtained using this technique. To overcome the H.sub.2 O.sub.2 dependence of the prior art, Mandle uses the large chemiluminescent reservoir of energy obtained in the oxalate system chemistry by adding a suitable quantity of hydrogen peroxide and oxalate.
In U.S. Pat. No. 4,396,579, Schroeder discusses that the mechanism of organic chemiluminescence in solution involves three stages: (1) a first preliminary reaction to provide an intermediate; (2) an excitation step in which the chemical energy of the intermediate is converted into excitation energy; and (3) emission of light from the excited product formed in the chemical reaction.
U.S. Pat. No. 4,647,532, Wantanabe, et al. relates to methods for determining hydrogen peroxide by chemiluminescence where a non-fluorescent substance and hydrogen peroxide are reacted in the presence of an oxidizing catalyst which produces a fluorescent substance and water. Thereafter, the fluorescent substance reacts with an oxalic acid ester in the presence of hydrogen peroxide to produce light. The oxidizable non-fluorescent substance used in the Wantanabe process are related to fluorescein and its derivatives.
In U.S. Pat. No. 4,994,377 (Nakamura, et al.), 1,5-anhydroglucitol is assayed by monitoring the hydrogen peroxide and in U.S. Pat. No. 5,093,238 (Yamashoji, et al.) determine the density or activity of viable cells by incubating the viable cells in the presence of a quinone whose reduced form reduces dissolved oxygen resulting in the formation of hydrogen peroxide. The hydrogen peroxide is reacted with a chemiluminiscent reagent in the presence of a fluorescent substance to cause fluorescence.
In U.S. Pat. No. 5,238,610 to Thompson a peroxy oxalate chemiluminiscent detection method is proposed which is more suitable for aqueous systems and relies upon a microemulsion. The fluorescent compounds used in the Thompson system are anthracene, napthacene, naphthalene, aminopyrine, dansyl amino acids, fluorescein and rhodamine derivatives. The drawback of calorimetric analysis is the necessity to monitor at different wavelengths (either in the visible or ultraviolet range) depending on the nature of the analyte.
A number of clinically important diagnostic tests are based on enzyme-coupled conversions involving the cofactors NAD.sup.+ /NADH, NADP.sup.+ /NADPH. These detection methods are spectrophotometric in nature, wherein the amount of reduced cofactor produced or consumed is directly proportional to the quantity of analyte. Colorimetric methods where one product of a particular transformation is hued (e.g. p-nitrophenol), or as with hydrogen peroxide, is enzymatically coupled to chromogenic agents (o-dianisidine, 4-aminoantipyrine) have been used. The resulting dye, i.e., quinoneimine dyes, are detected using spectrophotometric techniques.
In contrast to calorimetric methods, electrochemiluminescence (ECL) exploits the highly sensitive light-emitting property of a luminophore, such as tris(2,2'-bipyridyl)ruthenium(II), Ru(bpy).sub.3.sup.2+, and the strong reductive nature of an amine such as tripropylamine (TPrA). The use of luminescent metal labels for ECL detection is discussed in U.S. Pat. No. 5,221,605 to Bard, et al. incorporated herein by reference. Bard, et al. refer to the Mandle patent discussed above and indicates that Mandle discloses the use of chemiluminescent labels in amino acid chemical applications where the labels are excited into a luminescent state by chemical means, such as by reaction of the label with H.sub.2 O.sub.2 and an oxalate. In these systems, H.sub.2 O.sub.2 oxidatively converts the oxalate into a high energy derivative, which then excites the label. Bard, et al. express the opinion that the Mandle system, in principle, should work with any luminescent material that is stable in the oxidizing condition of the assay and can be excited by the high energy oxalate derivative. Unfortunately, this very versatility is a source of a major limitation of the technique because "typical biological fluids containing the analyte of interest also contain a large number of potentially luminescent substances that can cause high background levels of luminescence." Bard, et al. also indicate that the work of Rubenstein and Bard (1981), "Electrogenerated chemiluminescence." 37. Aqueous ECL Systems based on Ru(2,2 bipyridine).sub.3.sup.2+, an oxalate or organic acids, that demonstrates that bright orange chemiluminescence can be based on the aqueous reaction of chemically generated or electrogenerated Ru (bipyridine).sub.3.sup.3+ (where "bpy" represents a bipyridyl ligand) with strong reductants produced as intermediates in the oxidation of oxalate ions or other organic acids. However, Bard, et al. do not suggest a hydrogen peroxide-oxalate ECL system.
The intensely luminescent Ru(bpy).sub.3.sup.2+ can be used to quantify other systems including amines, amino acids and proteins. Prior methods for coupling ECL to enzymatic reactions are limited to those using nicotinamide adenine dinucleotide (NADH) cofactor-linked systems. Unfortunately, this technique limits the types of analytes which may be assayed. Prior ECL assays have used oxalate-based ECL, but have not been used to identify H.sub.2 O.sub.2, alone or from H.sub.2 O.sub.2 producing and consuming reactions.
A luminol-dependent chemiluminescence reaction has been described (Vilim, V. and Wilhelm, J. Free Radic Biol Med 6(6):623-629 (1989)) for detection of various oxygen species, such as superoxide anions, hydrogen peroxide, and hydroxyl radicals. Although the luminol-dependent chemiluminescence reaction is able to detect oxygen species, the reaction has little ability to discriminate between the various oxygen or radical species.
Thus a need exists for a reaction system that is not wavelength dependent, such as prior art colorimetric techniques, or limited to reactions based upon nicotinamide adenine dinucleotide (NADH) cofactor-linked systems. The current invention, as described hereinafter, provides a system for analyzing a wider variety of analytes to be assayed by using ECL.
Thus, a further need exists for a reaction system that is able to detect the presence of hydrogen peroxide and distinguish hydrogen peroxide from other oxygen species.